The role of NO in apoptosis signaling has been one of the most intensely studied topics in the past few years. However, published results are confusing and controversial. We have recently demonstrated that NO by itself exerts significant anti-apoptotic effect, whereas its secondary reaction products (e.g., peroxynitrite) are pro-apoptotic. Accumulating evidence suggests that protein nitration is a critical post-translational modification and myocardial ischemia/reperfusion (MI/R) causes significant protein nitration. Moreover, our preliminary experiments demonstrated for the first time that in vitro exposure of cultured cardiomyocytes to a pathologically relevant concentration of peroxynitrite or in vivo MI/R results in nitrative inactivation of thioredoxin, a novel anti-oxidant and anti-apoptotic protein, as well as thioredoxin reductase, the exclusive enzyme responsible for thioredoxin reduction and reactivation. Our long-term goals are to uncover the mechanisms responsible for myocardial apoptosis after MI/R, and to search for the optimal therapeutic strategies that will reduce myocardial reperfusion injury. The overall hypothesis to be tested in the present grant application is that thioredoxin/thioredoxin reductase nitration plays a causative role in myocardial apoptosis signaling, and that inhibiting thioredoxin/thioredoxin reductase nitration may be a novel therapeutic strategy to reduce MI/R injury. To test this hypothesis, we will address the following specific aims using both in vitro and in vivo experimental models: Specific Aim 1. To establish a causative link between thioredoxin/thioredoxin reductase nitration and apoptosis in cultured adult cardiomyocytes treated with peroxynitrite;Specific Aim 2. To determine the downstream signaling mechanisms by which nitration of thioredoxin/thioredoxin reductase leads to apoptotic cardiomyocyte death;Specific Aim 3. To identify molecular/cellular sources that are responsible for thioredoxin/thioredoxin reductase nitration after MI/R in vivo;and Specific Aim 4. To test the hypothesis that anti-nitration interventions may block thioredoxin/ thioredoxin reductase nitration and thus reduce myocardial infarction and improve myocardial functional recovery after MI/R. Ischemic heart disease remains to be the number 1 killer in the USA. Clarifying the mechanisms responsible for cell death after Ml and identifying novel therapeutic strategies will help to reduce ischemic disease related death in this country.